Detection of mycobacteria by multiplex nucleic acid amplification

ABSTRACT

Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 ant 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.

This application is a continuation-in-part of patent application U.S.Ser. No. 08/073,197 filed Jun. 4, 1993, now U.S. Pat. No. 5,422,252, acontinuation-in-part of patent application U.S. Ser. No. 08/058,648filed May 5, 1993, now U.S. Pat. No. 5,348,980, and acontinuation-in-part of patent application U.S. Ser. No. 08/060,842filed May 11, 1993, now abandoned, the disclosures of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid amplification anddetection and/or identification of microorganisms using nucleic acidamplification.

BACKGROUND OF THE INVENTION

In vitro nucleic acid amplification techniques have provided powerfultools for detection and analysis of small amounts of nucleic acids. Theextreme sensitivity of such methods has lead to attempts to develop themfor diagnosis of infectious and genetic diseases, isolation of genes foranalysis, and detection of specific nucleic acids as in forensicmedicine.

In general, diagnosis and screening for specific nucleic acids usingnucleic acid amplification techniques has been limited by the necessityof amplifying a single target sequence at a time. In instances where anyof multiple possible nucleic acid sequences may be present (e.g.,infectious disease diagnosis), performing multiple separate assays bythis procedure is cumbersome and time-consuming. U.S. Pat. Nos.4,683,195; 4,683,202 and 4,800,159 describe the PCR. Although theseinventors state that multiple sequences may be detected, no procedurefor amplifying multiple target sequences simultaneously is disclosed.When multiple target sequences are amplified, it is by sequentiallyamplifying single targets in separate PCRs. In fact, when multiple pairsof primers directed to different target sequences are added to a singlePCR, the reaction produces unacceptably high levels of nonspecificamplification and background. An improvement on the PCR which reportedlyallows simultaneous amplification of multiple target sequences isdescribed in published European Patent Application No. 0 364 255. Thisis referred to as multiplex DNA amplification. In this method, multiplepairs of primers are added to the nucleic acid containing the targetsequences. Each primer pair hybridizes to a different selected targetsequence, which is subsequently amplified in a temperature-cyclingreaction similar to PCR. Adaptation of PCR to footprinting is taught byP. R. Mueller and B. Wold (1989. Science 246, 780-786). Forfootprinting, a common oligonucleotide sequence is ligated to the uniqueend of each fragment of the footprint ladder. The fragments aresimultaneously amplified using a primer complementary to the commonsequence and a primer complementary to the known sequence of the fixedend.

In most cases, nucleic acid amplification techniques have been used toproduce qualitative results in diagnostic assays. However, there hasbeen great interest in developing methods for nucleic acid amplificationwhich are not only capable of detecting the presence or absence of atarget sequence, but which can also quantitate the amount of targetsequence initially present. Internal control sequences have been used inthe PCR in an attempt to produce such quantitative results. Parentapplication U.S. Ser. No. 08/058,648 discloses internal controlssequences useful in isothermal nucleic acid amplification reactions forquantitating target sequence as well as determining the amplificationactivity of the sample (i.e., efficacy--whether or not the sampleinhibits the amplification reaction, thus producing a false negativeresult).

Certain PCRs which employ internal controls select internal controlsequences which can be amplified by the same primers as the targetsequence. See, for example, WO 93/02215 and WO 92/11273. In the PCR, theamplified target and control sequences may distinguished by differentfragment lengths as the rate of the PCR is known to be relativelyunaffected by the length of the target and does not significantly affectamplification efficiency. EP 0 525 882 describes a method forquantifying a target nucleic acid in a Nucleic Acid Sequence BasedAmplification (NASBA) reaction by competitive amplification of thetarget nucleic acid and a mutant sequence. The method is performed witha fixed amount of sample and a dilution series of mutant sequence. Theanalysis involves determining the amount of added mutant sequence whichreduces the signal from the target sequence by 50%, i.e., the point atwhich the mutant sequence and target sequence are present in equalamounts. To produce accurate quantification, the amplification reactionsdescribed in EP 0 525 882 must be allowed to continue until at least oneof the reagents is essentially exhausted, i.e., into thepost-exponential phase of the reaction where competition for limitedreagents can occur. Furthermore, the calculations are accurate only whentwo reactions are competing for reagents--the target amplification andthe mutant sequence amplification. The results are therefore notreliable when a third reaction, such as background amplification, isoccurring. As essentially all amplification reactions include somedegree of background amplification, the EP 0 525 882 quantifying methodis only accurate for a high level of target sequence. At low targetlevels, competing background amplification reactions would significantlyinterfere with the accuracy of the calculations. Because it relies onamplifying various dilutions of the mutant sequence with the target, theEP 0 525 882 method is also susceptible to tube-to-tube variations inthe amount of mutant and target sequence. Even small differences in theamount of target sequence or slight inaccuracies in the dilutions ofmutant sequence between tubes are exponentially amplified in thesubsequent amplification reaction and are reflected in thequantification calculations.

In contrast, the method of U.S. Ser. No. 08/058,648 does not requirecompetition between control and target sequences for reagents nor doesit require that the reaction go into the post-exponential phase. It isaccurate in both the exponential and post-exponential phases of theamplification reaction. The ratio of target/control sequence istherefore not adversely affected by background amplification reactionswhich may be occurring and remains the same regardless of the extent ofbackground reaction. The result can therefore be obtained earlier in theamplification reaction and variability is reduced by the use of a singletarget/control coamplification reaction rather than a series ofreactions.

Previously reported multiplex nucleic acid amplification methods requirea separate internal control sequence matched to each target to beamplified because each target is amplified using a different pair ofprimers (i.e., a control sequence for each primer pair). Prior to thepresent invention it was not possible to use a single internal controlsequence to monitor or quantitate multiple targets in multiplex nucleicacid amplification reactions. It is therefore a feature of the instantadapter-mediated multiplex amplification methods that the single pair ofprimers required for multiplex amplification makes it possible for thefirst time to use a single internal control sequence to monitor orquantitate amplification of the multiple targets.

The Mycobacteria are a genus of bacteria which are acid-fast,non-motile, gram-positive rods. The genus comprises several specieswhich include, but are not limited to, Mycobacterium africanum, M.avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae,M. intracellulare, M. kansasii, M. microti, M. scrofulaceum, M.paratuberculosis and M. tuberculosis. Certain of these organisms are thecausative agents of disease. For the first time since 1953, cases ofmycobacterial infections are increasing in the United States. Ofparticular concern is tuberculosis, the etiological agent of which is M.tuberculosis. Many of these new cases are related to the AIDS epidemic,which provides an immune compromised population which is particularlysusceptible to infection by Mycobacteria. Other mycobacterial infectionsare also increasing as a result of the increase in available immunecompromised patients. Mycobacterium avium, Mycobacterium kansasii andother non-tuberculosis mycobacteria are found as opportunistic pathogensin HIV infected and other immune compromised patients.

At the present time the diagnosis of mycobacterial infections isdependent on acid-fast staining and cultivation of the organism,followed by biochemical assays. These procedures are time-consuming, anda typical diagnosis using conventional culture methods can take as longas six weeks. Automated culturing systems such as the BACTEC™ system(Becton Dickinson Microbiology Systems, Sparks, Md.) can decrease thetime for diagnosis to one to two weeks. However, there is still a needto reduce the time required for diagnosing Mycobacterial infections toless than a week, preferably to about one day. Oligonucleotide probebased assays such as Southern hybridizations or dot blots are capable ofreturning a rapid result (i.e., in one day or less). Assays based onamplification of nucleic adds may provide even more rapid results, oftenwithin hours. For diagnosis of Mycobacterial infections such methodswould require an oligonucleotide probe or primer which is specific forthe genus of Mycobacteria or specific for a particular mycobacterialspecies if specific identification of the organism is desired.

SUMMARY OF THE INVENTION

It has now been discovered that the primers disclosed in U.S. Ser. No.08/073,197 to exemplify adapter-mediated multiplex amplification of theIS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the16S ribosomal gene of Mycobacterium tuberculosis are also useful forsimultaneously detecting and/or identifying species of the M.tuberculosis complex and other clinically relevant Mycobacterium speciesby nucleic acid amplification. The inventive methods use multiplexStrand Displacement Amplification (SDA) in a single amplificationreaction which is capable of simultaneously identifying M. tuberculosisand providing a screen for substantially all of the clinically relevantspecies of Mycobacteria. SDA is capable of amplifying two target DNAsequences 10⁸ -fold during a single incubation at a constanttemperature.

In a particularly preferred embodiment, the amplification reactionfurther includes an internal control sequence as described in U.S. Ser.No. 08/058,648. This internal control sequence is co-amplified with thetwo target sequences in a multiplex amplification protocol employing asingle pair of amplification primers for simultaneous amplification ofthe IS6110, 16S and internal control targets (triplex amplification). Inthis embodiment the assay, in a single amplification reaction, providesmeans for quantitating target or determining sample amplificationactivity and detecting/identifying clinically relevant Mycobacteriawhich may be present. The simultaneous amplification of genus- andspecies-specific Mycobacterium sequences with a single internal controlsequence exemplifies the broader applicability of the disclosed methodsfor adapter-mediated multiplex amplification of multiple targets and asingle internal control sequence using the same primer pair.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the method of the invention used forcoamplification of two target sequences in Example 1, Example 2 andExample 3.

FIG. 2 shows sequence alignments for the Mycobacterium genus targetsequence in the 16S ribosomal genes of various Mycobacterium species,including illustration of the primer binding sites.

FIG. 3 is an autoradiograph showing the results of the experiment inExample 1.

FIG. 4 is an autoradiograph showing the results of the experiment inExample 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for simultaneous amplification ofmultiple target sequences by sequence specific hybridization of primers,particularly by SDA (multiplex SDA). The methods use a single pair ofamplification primers or a single SDA amplification primer to coamplifythe multiple target sequences. This is accomplished by appending adefined adapter sequence to the targets and amplifying by primerextension. The inventive methods are referred to herein as"adapter-mediated multiplexing." This is in contrast to "conventionalmultiplexing" in which multiple pairs of target-specific primers areused to coamplify the multiple targets without addition of adaptersequences.

The following terms are defined herein as follows:

An amplification primer is a primer for amplification of a targetsequence by sequence specific hybridization. For SDA, the 3' end of theamplification primer (the target binding sequence) hybridizes at the 3'end of the target sequence and comprises a recognition site for arestriction enzyme near its 5' end. The recognition site is for arestriction enzyme which will nick one strand of a DNA duplex when therecognition site is hemimodified, as described by Walker, et al. (1992.PNAS 89, 392-396 and Nucleic Acids Res. 20, 1691-1696) and in U.S. Ser.No. 07/819,358, filed Jan. 9, 1992 (the disclosure of which is herebyincorporated by reference). A hemimodified recognition site is a doublestranded recognition site for a restriction enzyme in which one strandcontains at least one derivatized nucleotide which prevents cutting ofthat strand by the restriction enzyme. The other strand of thehemimodified recognition site does not contain derivatized nucleotidesat the cleavage site and is nicked by the restriction enzyme. Thepreferred hemimodified recognition sites are hemiphosphorothioatedrecognition sites for the restriction enzymes HincII, HindII, AvaI, NciIand Fnu4HI. For the majority of the SDA reaction, the amplificationprimer is responsible for exponential amplification of the targetsequence.

An adapter primer is an oligonucleotide which has a sequence at its 3'end (the target binding sequence) for hybridizing to the targetsequence. At the 5' end of the adapter primer is an adapter sequence.The adapter sequence may be a sequence which is substantially identicalto the 3' end of one of the amplification primers or it may be anydefined sequence for which amplification primers with complementarytarget binding sequences can be prepared.

A bumper primer is a primer which anneals to a target sequence upstreamof either an adapter or amplification primer, such that extension of thebumper primer displaces the downstream primer and its extension product.Extension of bumper primers is one method for displacing the extensionproducts of adapter and amplification primers, but heating is alsosuitable.

Identical sequences will hybridize to the same complementary nucleotidesequence. Substantially identical sequences are sufficiently similar intheir nucleotide sequence that they also hybridize to the samenucleotide sequence.

The terms target or target sequence refer to nucleic acid sequences tobe amplified. These include the original nucleic acid sequence to beamplified and its complementary second strand (prior to addition ofadapter sequences), either strand of an adapter-modified copy of theoriginal sequence as described herein, and either strand of a copy ofthe original sequence which is an intermediate product of the reactionsin which adapter sequences are appended to the original sequence.

Species-specific amplification of a target sequence refers toamplification of a target sequence in the species of Mycobacteriaclassified as members of the Mycobacterium tuberculosis complex, butlittle or no amplification in non-M.tb complex Mycobacteria.

Genus-specific amplification of a target sequence refers toamplification of a target sequence in substantially all of theclinically relevant species of Mycobacteria, but little or noamplification in similar non-Mycobacterium species.

In the adapter-mediated multiplexing of the invention, adapter sequencesare appended to the ends of target sequences by means of adapter primersand a series of extension and strand displacement steps. FIG. 1illustrates one embodiment of the present invention in which two targetsequences are co-amplified using a single pair of amplification primers.One of the two target sequences is M.tb complex-specific (e.g., IS6110)and the other target sequence is specific for all genera ofMycobacterium (M.g--e.g., the 16S gene). Modification of only one strandof each target sequence is illustrated for clarity. In this embodiment,one end of each target strand is modified by appending to it a sequencesubstantially identical to a terminal segment of the other target. Theother end of each target strand remains unmodified and retains itsoriginal complementarity to one member of the amplification primer pair.As detailed below, the resulting modified targets can then both beamplified by a single pair of amplification primers, one member of thepair being complementary to one of the two original target sequences andthe other member of the pair being complementary to the other of the twooriginal target sequences. For the first (M.tb complex-specific) target(M.tb Target), an M.tb-specific amplification primer (S_(tb)) ishybridized to the 3' end of the target sequence and extended withpolymerase. The nicking enzyme recognition site of the amplificationprimer is depicted in FIG. 1 as a raised portion of the primer. Theresulting extension product is displaced by extension of a bumper primer(B_(tb1)) which hybridizes to the target upstream from S_(tb). Thedisplaced S_(tb) extension product (S_(tb) -ext) is hybridized to anadapter primer (A_(tb)) which binds to S_(tb) -ext at the 3' end of thecomplement of the original target sequence. The 5' end of A_(tb)comprises the adapter sequence (solid portion), which is substantiallyidentical to the target binding sequence at the 3' end of S_(g), anamplification primer which specifically binds to the second,Mycobacterium genus-specific target (M.g Target). Extension of A_(tb)and displacement of the A_(tb) extension product (A_(tb) -ext) producesa single stranded copy of the M.tb target sequence with a nicking enzymerecognition site and the M.tb target sequence at its 3' end and theS_(g) target binding sequence at its 5' end.

The second target (M.g Target) is treated similarly, first binding andextending an M.g-specific amplification primer (S_(g)), then hybridizingan adapter primer (A_(g)) to the extension product (S_(g) -ext). S_(g)hybridizes to the M.g target at a 3' terminal segment of the targetwhich is complementary to both the target binding sequence of S_(g) andthe adapter sequence of A_(tb). The 3' end of adapter primer A_(g)hybridizes at the 3' end of the complement of the original target andthe 5' end of A_(g) (open portion) is substantially identical to thetarget binding sequence of S_(tb). Extension and displacement of theA_(g) extension product (A_(g) -ext) produces a copy of the secondtarget sequence with a nicking enzyme recognition site (raised portion)and the M.g target sequence at its 3' end and the S_(tb) target bindingsequence at its 5' end. The two adapter-modified copies of the targetsequences are amplifiable by SDA using only the S_(tb) and S_(g)amplification primers already present in the reaction. To begin SDA,A_(tb) -ext and A_(g) -ext hybridize to their respective amplificationprimers, which are extended to produce the complement of the modifiedstrand (i.e., extension of S_(tb) on the M.tb modified strand andextension of S_(g) on the M.g modified strand), including the complementof the adapter sequence at the 3' end. After nicking and displacement,the amplification primer of the opposite target can then bind to the 3'end of this extension product (i.e., S_(g) to the M.tb-derived strandand S_(tb) to the M.g-derived strand) and is extended to produce afragment with a nicking enzyme recognition site at each end. Thisfragment is amplified by conventional SD as described by Walker, et al.,supra.

The double stranded reaction products which are produced afterdisplacement of A_(tb) -ext and A_(g) -ext may also participate in areaction loop which generates additional copies of A_(tb) -ext and A_(g)-ext. Nicking the restriction enzyme recognition site of the bottomstrand, extending with polymerase and displacing the bottom strandproduces targets which are similar to S_(tb) -ext and S_(g) -ext butwith half of a restriction enzyme recognition site at the 5' end. Theadapter primers can bind to these fragments and can be extended anddisplaced to produce additional copies of A_(tb) -ext and A_(g) -ext(also with half of a restriction enzyme recognition site at the 5' end)which enter the SDA reaction cycle as described above.

FIG. 1 depicts the generation of modified targets from only one of thetwo complementary strands normally present fix each target sequence.Processes similar to those shown also originate from the second strandof each target. In the case of the second strand, however, the order ofbinding and extension of the primers is reversed. The adapter primersfirst bind directly to the target second strand and are extended on thattemplate. After its subsequent displacement, the resulting adapterextension product hybridizes to the amplification primer, which is inturn extended and displaced to give a product containing the originalsecond strand target sequence with a recognition site for a nickingrestriction enzyme at its 5' end and a sequence complementary to theadapter sequence at its 3' end. This modified fragment entersconventional SDA amplification by binding and extension of theamplification primer specific for the opposite target (i.e., S_(g) bindsto the M.tb-derived strand and S_(tb) binds to the M.g-derived strand),producing a fragment for each target second strand with a nicking enzymerecognition site at each end.

All of the reaction steps involved in appending the adapter sequencesand amplifying the target may occur concurrently in a single reactionmixture. That is, once adapter sequences are appended to a targetmolecule, amplification of that target molecule can take place withinthe same reaction mixture prior to appending of the adapter sequences toany other target molecules present and without isolation of the modifiedtarget. Reaction conditions for the methods of the invention areessentially as described by Walker, et al., supra, for SDA, with somemodifications. First, the initial reaction mix contains both theamplification primers and the adapter primers as well as the target DNA.In addition, the amplification primers are preferably present in about10-fold excess over the adapter primers and about 20-fold excess overthe bumper primers. The concentration of bumper primers is not critical,but will generally be less than the concentration used in conventionalSDA. However, like conventional SDA, the nicking restriction enzyme andexo⁻ klenow polymerase are added after heat denaturation of the targetDNA and annealing of the primers. After denaturation of the target DNA,annealing of the primers and addition of polymerase, the processes ofappending the adapter sequences and amplification proceed automaticallyin a single reaction mixture without further intervention by thepractitioner. That is, after adapter sequences are appended, a modifiedtarget sequence automatically enters the SDA reaction cycle.

The complete nucleotide sequence of the IS6110 insertion element hasbeen described by Thierry, et al. (1990. Nucleic Acids Res. 18, 188).The methods of the invention provide primers which amplify a targetsequence within the IS6110 insertion element which is present in thespecies of the Mycobacterium complex (M. tuberculosis, M. bovis, M.bovis BCG, M. africanum and M. microti). The primers are complementaryto nucleotides 972-984 of IS6110 and support little or no amplificationof target in Mycobacterium species other than the M.tb complex or innon-Mycobacterium species. These primers are defined herein asspecies-specific primers.

Alignment of the 16S ribosomal genes of various Mycobacterium species,as shown in FIG. 2, was used to design genus-specific primers fornucleic acid amplification which would amplify a target in substantiallyall of the clinically relevant species of Mycobacteria but not innon-Mycobacterium species. The selected M. tuberculosis sequence atnucleotide positions 507-603 is identical to the sequences in M. bovis,M. bovis BCG, M. avium, M. intracellulare, M. kansasii, M. gastri, M.paratuberculosis, M. malmoense, M. szulgai, M. gordonae, M. leprae, M.ulcerans, M. asiaticum and M. scrofulaceum. For some species in thesequence database, an undetermined nucleotide is listed at position 587or 588, but neither of these positions involve primer binding. M.terrae, M. chelonae, M. fortuitum and our strain of M. marinum vary fromthis common sequence at three nucleotide positions. The sequence of ourstrain of M. marinum was unexpected, as the GENBANK sequence for thisorganism was reportedly identical to M. bovis, not M. terrae. M.flavescens, M. xenopi and M. genavense exhibit more extensive sequencevariance. This target sequence diverges significantly from the M.tbsequence in organisms which are otherwise generally similar toMycobacteria. In spite of the sequence variability among Mycobacteria,however, it was found that the primers designed to amplify the targetsequence as shown in FIG. 2 specifically amplified a target present inthe clinically relevant genera of Mycobacteria but not in other similarnon-Mycobacterium species. These primers are defined herein asgenus-specific primers.

Simultaneous amplification of the M.tb species-specific IS6110 sequenceand the genus-specific 16S sequence in a multiplex SDA reaction allowsrapid detection and/or identification of M.tb organisms and otherclinically relevant Mycobacterium species in a single reaction.Detection of amplified IS6110 and 16S targets is indicative of thepresence of an M. tuberculosis complex organism in the sample. Detectionof only 16S targets indicates the presence of non-M.tb complexMycobacteria. As results can be obtained within a day using nucleic acidamplification methods, it is no longer necessary to wait six weeks forculture results before reaching a decision that a sample is negative forclinically relevant Mycobacteria.

In a first embodiment, IS6110 and 16S target sequences are co-amplifiedby adapter-mediated multiplex SDA using the genus-specific andspecies-specific primers of the invention. In an alternative preferredembodiment, the adapter-mediated multiplex SDA reaction further includesan internal control sequence as, described in U.S. Ser. No. 08/058,648.The preferred internal control sequence for use with the genus-specificand species-specific primers of the following Examples is (SEQ ID NO:12). SEQ ID NO: 12 contains the core sequence of SEQ ID NO: 1 of U.S.Ser. No. 08/058,648 and an amplification primer binding sequence at eachend to facilitate co-amplification using a single pair of primers astaught in U.S. Ser. No. 08/073,197. The amplification primer bindingsequences of the internal control sequence are complementary to thetarget binding sequences of the amplification primers.

It will be apparent to one skilled in the art from the foregoingdisclosure that either the IS6110 or 16S sequences may be amplifiedalone in a conventional SDA reaction. For example, for M.tb detectiononly the 16S sequence may be amplified according to Walker, et al. usingS_(g) and S_(tb) with the A_(g) adapter primer. Alternatively, when onlygenus-specific detection is desired, the 16S sequence may be amplifiedusing S_(g) and a A_(g) modified to function as an amplification primer,i.e., replacing the M.tb adapter sequence with a HincII restrictionenzyme recognition site. Similarly, the IS6110 target sequence alone maybe amplified using S_(g) and S_(tb) with the A_(tb) adapter primer, orusing S_(tb) and an A_(tb) adapter primer modified to function as anamplification primer by replacing the M.g adapter sequence with a HincIIrestriction enzyme recognition site.

The amplification products of the IS6110, 16S and internal controltarget sequences may be detected by hybridization to oligonucleotideprobes tagged with a detectable label, each one of three probesspecifically hybridizing to one of the targets. If the target-specificand control-specific probes are hybridized simultaneously to theamplification products, the labels should be separately identifiable tofacilitate distinguishing the respective amounts of control and target.Otherwise, separate aliquots of the amplification reaction may beseparately hybridized to target-specific and control-specific probestagged with the same label. The detectable label may be conjugated tothe probe after it is synthesized or it may be incorporated into theprobe during synthesis, for example in the form of a label-derivatizednucleotide. Such labels are known in the art and include directly andindirectly detectable labels. Directly detectable labels produce asignal without further chemical reaction and include such labels asfluorochromes, radioisotopes and dyes. Indirectly detectable labelsrequire further chemical reaction or addition of reagents to produce thedetectable signal. These include, for example, enzymes such ashorseradish peroxidase and alkaline phosphatase, ligands such as biotinwhich are detected by binding to label-conjugated avidin, andchemiluminescent molecules. The probes may be hybridized to theirrespective amplification products in solution, on gels, or on solidsupports. Following hybridization, the signals from the associatedlabels are developed, detected and separately quantitated using methodsappropriate for the selected label and hybridization protocol. Theamount of signal detected for each amplification product is a reflectionof the amount present.

One preferred method for detecting the target and control amplificationproducts is by polymerase extension of a primer specifically hybridizedto the target or control sequence. The primer is labeled as describedabove, preferably with a radioisotope, so that the label of the primeris incorporated into the extended reaction product. This method isdescribed in more detail by Walker, et al. (1992 ) Nuc. Acids Res. andPNAS, supra. A second preferred method for detecting amplified targetand control sequences is a chemiluminescent method in which amplifiedproducts are detected using a biotiniylated capture oligodeoxynucleotideprobe and an enzyme-conjugated detector oligodeoxynucleotide probe asilllustrated in Example 3. After hybridization of these two probes todifferent sites on an amplified target sequence, the complex is capturedon a streptavidin-coated microtiter plate, and the chemiluminescentsignal is developed and read in a luminometer. This detection method canbe performed in less than two hours and is sensitive enough to detect asfew as one pre-amplification target sequence.

SDA reactions employing the primers of the invention may incorporatethymine as taught by Walker, et al., supra, or they may wholly orpartially substitute 2'-deoxyuridine 5'-triphosphate for TTP in thereaction (as shown in the instant Examples) as a means for reducingcross-contamination of subsequent SDA reactions as taught in U.S. Ser.No. 08/060,842. dU is incorporated into amplification products of bothtarget and control sequences and can be excised by treatment with uracilDNA glycosylase (UDG). These abasic sites render the amplificationproduct unamplifiable in subsequent SDAs. The internal control sequenceas initially synthesized may also incorporate dU in place of thymine toprevent its amplification in subsequent SDAs. For example, SEQ ID NO: 12is shown in the attached Sequence Listing as containing thymine but itmay also consist of the corresponding sequence in which thymine isreplaced wholly or paratially by dU. UDG may be inactivated by uracilDNA glycosylase inhibitor (Ugi) prior to performing the subsequentamplification to prevent excision of dU in newly-formed amplificationproducts.

As certain of the primers and probes disclosed herein and exemplified inthe following Examples are identical in sequence to primers and probespreviously disclosed in the parent applications, the followingcorrespondence of Sequence ID Nos. is provided for clarity:

    ______________________________________                                        SEQ ID  CORRESPONDING SEQ                                                     NO: IN- ID NO: IN-                                                            Instant U.S. Ser. No.                                                                            U.S. Ser. No.                                              Application                                                                           08/073,197 08/058,648 FUNCTION                                        ______________________________________                                         1      2          None       IS6110 Amplification                                                          Primer                                           2      10         None       IS6110 Adapter                                                                Primer                                           3      5          None       IS6110 Bumper                                                                 Primer                                           4      6          None       IS6110 Bumper                                                                 Primer                                           5      3          None       16S Amplification                                                             Primer                                           6      9          None       16S Adapter Primer                               7      7          None       16S Bumper Primer                                8      8          None       16S Bumper Primer                                9      None       None       M.tb Detector                                                                 (Primer Ext.)                                   10      None       None       16S Detector (Primer                                                          Ext.)                                           11      None       None       16S Detector (Primer                                                          Ext.)                                           12      None       None       Internal Control                                                              Sequence                                        13      None       6          IS6110 Capture                                  14      None       7          IS6110 Detector                                                               (Hyb.)                                          15      None       4          Internal Control                                                              Capture                                         16      None       5          Int. Control Detector                                                         (Hyb.)                                          17      None       None       16S Capture                                     18      None       None       16S Detector                                                                  (Hybridization)                                 19      None       None       16S Detector -fc                                                              (Hyb.)                                          ______________________________________                                    

The primers and/or probes for performing the assay methods of theinvention may be packaged in the form of a diagnostic kit forsimultaneous genus-specific and species-specific amplification ofMycobacterium DNA. The kits may comprise the amplification, adapter andbumper primers for genus-specific and species-specific amplification ofMycobacterium DNA as well as the reagents required for performing theSDA reaction (e.g., deoxynucleoside triphosphates, nicking restrictionenzymes, buffers, exo⁻ polymerase, etc.). The kits may furtheroptionally include the probes or primers useful for detecting andidentifying the amplification products and/or an internal controlsequence for co-amplification with the Mycobacterium target sequences.

EXAMPLE 1

This experimental example demonstrates coamplification of genus- andspecies-specific target nucleic acids using the amplification methodillustrated in FIG. 1. The first target was the IS6110 insertion elementof M. tuberculosis (target A). The second target was the 16S ribosomalgene of M. tuberculosis (target B). An amplification reaction was set upfor each of the following species: M. tuberculosis H37Rv (ATCC 27294),M. bovis (CDC 81), M. bovis-BCG (CDC34), M. avium (ATCC 25291), M.intracellulare (ATCC 13950), M. kansasii (LCDC 711), M. gastri (LCDC1301), M. fortuitum (LCDC 2801), M. paratuberculosis (LINDA), M.chelonae (TMC 1543), M. malmoense (CDC 93), M. szulgai (TMC 1328), M.flavescens (LCDC 2601), M. xenopi (LCDC 1901), M. terrae (TMC 1450), M.marinum (LCDC 801) and M. gordonae (TMC 1318). The "no target" samplecontained no bacterial target DNA. The "no SDA" sample represented acontrol reaction to which no amplification enzymes were added.

SDA was performed generally as described by Walker, et al., Nuc. AcidsRes., supra, substituting dUTP for TTP to allow removal of contaminatingamplicons. The final concentrations of components was 45 mM K_(i) PO₄,pH 7.5, 6 mM MgCl₂, 0.5 mM dUTP, 0.2 mM dGTP, 0.2 mM dCTP, 0.2 mMdATPαS, 0.1 mg/mL acetylated BSA, 12% (v/v) dimethylsulfoxide, 3% (v/v)glycerol (supplied by the stock solutions of exo⁻ klenow and HincII), 50ng human placental DNA, 2.5 units exo⁻ klenow (United StatesBiochemical, Cleveland, Ohio), 150 units HincII (New England Biolabs,Beverly, Mass.), and 1000 genomes (molecules) of the Mycobacteriumspecies being tested. Each sample contained two sets of four primers, asfollows: Set #1) 500 nM of SEQ ID NO: 1 (S_(tb) in FIG. 1), 50 nM of SEQID NO: 2 (A_(tb)), 25 nM of each of SEQ ID NO: 3 (B_(tb1)) and SEQ IDNO: 4 (B_(tb2)); Set #2) 500 nM of SEQ ID NO: 5 (S_(g) in FIG. 1); 50 nMof SEQ ID NO: 6 (A_(g)), 25 nM of each of SEQ ID NO: 7 (B_(g1)) and SEQID NO: 8 (B_(g2)).

The primers of Set #1 are for species-specific detection of the IS6110element. The IS6110 amplification primer (S_(tb)) is complementary tonucleotides 972-984 of the IS6110 sequence. The bumper primers in thisexample are complementary to nucleotides 954-966 and 1032-1044 of theIS6110 sequence respectively, however, other bumper primers which can behybridized and extended to displace the extension product of the Set #1amplification primer may be selected based on knowledge of the IS6110sequence. The primers of Set #2 are for genus-specific detection ofclinically relevant Mycobacteria.

Each 47 μL sample was assembled to contain all reagents except exo⁻klenow and HincII using 10X concentrated stock solutions of eachreagent. The MgCl₂ was added after addition and mixing of all otherreagents (except exo⁻ klenow and HincII to prevent precipitation whichoccurs when K_(i) PO₄, dimethylsulfoxide and MgCl₂ are mixed atconcentrations considerably higher than 45 mM, 12% (v/v) and 6 mM,respectively. The samples were then heated for 2 min. in a boiling waterbath to denature the Mycobacterial DNA. A precipitate was observedimmediately after removal from the boiling water bath. Incubating for 2min. at 40° C. and mixing on a vortex mixer redissolved the majority ofthe high temperature precipitate. Exo⁻ klenow (1 μL of a 2.5 units/μLstock solution) and HincII (2 μL of a 75 units/μL stock solution) wereadded for a total sample volume of 50 μL, and the samples were incubatedfor 2 hr. at 40° C.

Amplification products were detected by primer extension as described byWalker, et al., Nuc. Acids Res., supra, also substituting dUTP. A 5 μLaliquot of each sample was mixed with 5 μL of 45 mM K_(i) PO₄, pH 7.5, 6mM MgCl₂, 0.5 mM dUTP, 0.2 mM dGTP, 0.2 mM dCTP, 0.2 mM dATPαS, 0.1mg/mL acetylated BSA and 2 μL of a 5'-³² P detector probe stock solution(50 mM Tris-HCl, pH 8, 10 mM MgCl₂, 1 μM of each of the three 5'-³² Pdetector probes). The detector probe for the IS6110 target was SEQ IDNO: 9 (species-specific for M.tb complex). The detector probes for the16S target were SEQ ID NO: 10 and SEQ ID NO: 11. SEQ ID NO: 11corresponds to the 16S sequences of M. fortuitum, M. chelonae, M.terrae, M. marinum and M. flavescens ("fc" detector). SEQ ID NO: 10corresponds to the remaining non-M.tb complex species tested. The 12 μLsamples were then heated 1 min. in a boiling water bath. Afterincubating 2 min. at 37° C., 2 μL of 1 unit/μL of exo⁻ klenow were addedand the samples were incubated for 15 min. at 37° C., followed byaddition of 14 μL of 50% urea in 0.5X TBE.

Samples were heated for 1 min. at 95° C. and analyzed using 8%denaturing gel electrophoresis and autoradiography (Maniatis, et al.1982. Molecular Cloning: A Laboratory Manual Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). The results are shown in FIG. 3.Amplified IS6110 (M.tb target) is indicated by extension of the SEQ IDNO: 9 detector probe to a 41- and 63-mer. Amplified 16S (M.g target) isindicated by extension of the SEQ ID NO: 10 and SEQ ID NO: 11 detectorprobes to a 30- and 51-mer.

As shown in FIG. 3, positive IS6110 and 16S signals were obtained for M.tuberculosis, M. bovis and M. bovis-BCG, all of which are members of theM. tuberculosis complex. For M. tuberculosis, the IS6110 signals werestronger than the 16S signals because this organism contains about 10copies of the IS6110 element compared with a single copy of the 16Sribosomal gene. Roughly equivalent IS6110 and 16S signals were obtainedwith M. bovis and M. bovis-BCG because they contain one to two copies ofthe IS6110 element and a single copy of the 16S ribosomal gene. IS6110signals were not seen for any Mycobacterium species tested which was nota member of the M.tb complex.

16S signals only were detected for M. avium, M. intracellulare, M.kansasii, M. gastri, M. paratuberculosis, M. malmoense, M. szulgai andM. gordonae, which have 16S target sequences identical to that of M.tuberculosis. M. gordonae is not normally pathogenic, but is a commoncontaminant in clinical laboratories. It is therefore possible thatcontamination of a negative sample with M. gordonae would produce afalse positive result for the Mycobacterium genus in this test, as itdoes in culture-based tests. Relatively weak 16S signals were obtainedfor M. fortuitum, M. chelonae, M. marinum and M. terrae. It is possiblethat this result is related to a T--G mismatch which adapter SEQ ID NO:6 forms in these species (see FIG. 2). However, the same experimentperformed using a genus-specific adapter primer with a perfect matchstill produced a relatively weak signal, raising the possibility thatthe 16S gene of these species contains secondary structure whichattenuates amplification. The signal obtained for M. flavescens was asstrong as that for M. fortuitum despite an additional A--C mismatch atthe 3'-end of adapter SEQ ID NO: 6. No 16S signal was obtained for M.xenopi, possibly due to a poor match with the detector probes. Based onthese results, positive 16S signals would also be expected forMycobacterium species in addition to those tested (e.g., M.scrofulaceum, M. leprae, M. ulcerans, M. hemophilum and M. asiaticum),as the 16S sequences of these species also match that of M.tb.

EXAMPLE 2

This experimental example demonstrates the lack of cross-reactivity ofthe coamplification method with non-Mycobacteria. The amplifications andanalyses were performed as in Example 1, except that target DNA wastaken from either M. tuberculosis H37Rv (ATCC 27294) or the followingnon-Mycobacterium species: Corynebacteria diphtheriae (ATCC 11913),Corynebacteria xerosis (ATCC 373), Corynebacteria pseudodiphtheriticum(ATCC 10700), Nocardia asteroides (ATCC 3308), Nocardia brasiliensis(ATCC 19296), Nocardia orientalis (ATCC 19795), Streptomyces somaliensis(ATCC 33201), Streptomyces griseus (ATCC 10137), Streptomyces albus(ATCC 3004), Streptomyces gedanensis (ATCC 4880), Actinomyces israelii(ATCC 10049), Eubacterium lentum (ATCC 43055), Rhodococcus equi (ATCC6939), Rhodococcus rhodochrous (ATCC 13808), Propionibacterium acnes(ATCC 6919), Actinoplanes auranticolor (ATCC 15330), Streptosporangiumvirialbum (ATCC 33328), and Streptoverticillium alboverticillatum (ATCC29818). Different species of the same genus were pooled together in oneSDA sample as represented under the genus name. The number of M.tuberculosis genomes present in each SDA reaction is indicated in FIG.4. The non-Mycobacterium samples contained 10⁵ genome copies of eachindicated species. The "no target" sample did not contain bacterial DNA.The ³² -p bands corresponding to amplification products for the IS6110target (M. tuberculosis complex) and the 16S target (Mycobacteriumgenus) are indicated in FIG. 4.

All non-Mycobacterium organisms tested (10⁵ genomes) produced signalsweaker than the signal obtained for only 10 genomes of M. tuberculosis,even though the non-Mycobacterium organisms tested were generallysimilar to Mycobacterium.

EXAMPLE 3

This experimental example illustrates the embodiment of the invention inwhich an internal control sequence is co-amplified with the IS6110 and16S target sequences using the IS6110/16S amplification primer pair(triplex amplification). The amplifications were performed as in Example1 using M.tb DNA diluted to varying extents to assess the sensitivity ofthe detection methods. The amplification products were detected in asolid-phase chemiluminescent assay in which amplified target and controlsequences were captured on microwell plates by hybridization to animmobilized capture probe. hybridization was detected by sandwichhybridization of the captured target or control sequence to a detectorprobe labeled with alkaline phosphatase. The capture probes were labeledat either the 5' or 3' end with biotin and immobilized by binding tostreptavidin on the microwell plate. The detector probes were labeled ateither the 5' or 3' end with alkaline phosphatase (AP) and detected byenzymatic reaction with LUMIPHOS 530 (Lumigen, Inc., Detroit, Mich.).

Oligodeoxynucleotide capture probes with 5' biotinylation (SEQ ID NO: 13for the IS6110 target sequence and SEQ ID NO: 15 for the internalcontrol sequence) were synthesized as described in Example 2 of U.S.Ser. No. 08/058,648. Oligodeoxynucleotide detector probes 3' labeledwith AP (SEQ ID NO: 14 for IS6110 and SEQ ID NO: 16 for the internalcontrol) were also synthesized and labeled as in U.S. Ser. No.08/058,648.

Preparation of 3'-Biotinylated Capture Oligodeoxynucleotides

Oligodeoxynucleotide capture probes with 3' biotinylation weresynthesized as follows. SEQ ID NO: 17 (capture probe for the 16S targetsequence) was synthesized using a DNA synthesizer (Model 380B, AppliedBiosystems, Foster City, Calif.). 3'BIOTIN-ON CPG (controlled poreglass, Clonetech, Palo Ako, Calif.) was used to attach a biotin moietyat the 3' terminus of the oligodeoxynucleotide. Two additional biotinswere then added to the 3' terminus using BIOTIN-ON phosphoramiditereagent (also from Clonetech). The oligodeoxynucleotides were thenprepared using standard phosphoramidite reagents and cleaved from thesolid phase to give crude: 3'-biotinylated oligodeoxynucleotides.Purification was done by reverse phase High Pressure LiquidChromatography (HPLC) (Brownlee Lab Aquapore RP 300 Column--220×4.6 mm,C8 column 7 particle, 300 Å pore size) with a UV monitor at 254 nm and agradient of 14 to 44% Buffer B over one hour (Buffer B: 0.1MTriethylamine-Acetate pH 7 with 50% Acetonitrile; Buffer A: 0.1MTriethylamine-Acetate, pH 7) and a flow rate of 1 ml/minute.

Preparation of 5'-Alkaline Phosphatase Detector Oligodeoxynucleotides

Oligodeoxynucleotide detector probes for 5' labeling with alkalinephosphatase were synthesized from 5'-amino-oligodeoxynucleotidesprepared using a DNA synthesizer (Model 380B, Applied Biosystems, FosterCity, Calif.). SEQ ID NO: 19 is the detector probe for the 16S targetsequence in M. fortuitum, M. chelonae, M. terrae and M. flavescens ("fc"detector). SEQ ID NO: 18 is the detector probe for the 16S targetsequence in the remaining non-M.tb species of Mycobacteria tested. Thereagent AMINOLINK II (Applied Biosystems, Foster City, Calif.) was usedto place an amine group on the 5'-ends of the oligodeoxynucleotides forsubsequent conjugation with alkaline phosphatase as described above. Thecrude conjugates were dialyzed into 20 mM Tris pH 7.5 and concentratedusing a CENTRIPREP 30 (Amicon, Danvers, Mass.) to approximately 2 ml.The concentrated conjugated were then purified by HPLC using a DEAE-5PWcolumn (7.5 mm×7.5 cm) and a gradient of 0 to 66% Buffer B (Buffer B: 20mM Tris, 1M NaCl pH 7.5, Buffer A: 20 mM Tris pH 7.5) and a flow rate of1 ml/minute. Absorbance was monitored at 254 nm. The fractions werecollected, the activity of the conjugate was determined, and theconjugated were stored as described above.

Preparation of Coated Microwell Plates

Biotinylated bovine serum albumin (biotin*BSA) (Pierce, Rockford, Ill.)was diluted to 5 μg/ml in 50 mM carbonate pH 9.6 (Sigma, St. Louis, Mo.,prepared using autoclaved water) and was pipetted into each well (100μl/well) of Labsystems strip wells (Labsystems, Research Triangle Park,N.C.), and incubated at room temperature overnight. The plates werewashed twice (375 μl/wash) using FTA hemagglutination buffer pH 7.2(Becton Dickinson Microbiology Systems, Cockeysville, Md.) preparedusing autoclaved water. Streptavidin in hemagglutination buffer wasadded to the biotin*BSA coated wells (100 μl/well). Plates were coveredand incubated overnight at room temperature. Unbound streptavidin wasdiscarded by inversion of the wells and blocking buffer (300μl/well--hemagglutination buffer pH 7.2, 0.05% w/v BSA) was added. Theplates were covered and incubated overnight as above, the blockingbuffer was discarded by inversion of the wells. Plates were washed twicewith hemagglutination buffer (375 μl/well), then once usinghemagglutination buffer with 2% w/v trehalose (375 μl/well--Fluka,Ronkonkoma, N.Y.). Plates were dried for 1 hour at 37° C., sealed inmylar pouches with desiccant, and stored overnight at room temperatureprior to use. The plates were stored thereafter at 2°-8° C.

Microwell Assay Procedure

SDA was performed as in Example 1 on samples containing 0, 5, 10,20, 40and 80 copies of the M.tb genome. A "heat killed" sample was alsoincluded to assess the background luminescence generated by the detectorprobe. The heat killed sample was heated immediately upon beginning theSDA reaction to prevent amplification. Each reaction also contained 1000copies of the internal control sequence (SEQ ID NO: 12 in which thymineis replaced by dU). Forty-five μl of each completed SDA reaction wasdiluted into 180 μl of sterile water in a sterile siliconized tube. Thediluted SDA reactions were heated to 95° C. for 3 min. to denature theDNA. Tubes were cooled for 5 min. at room temperature and then 50 μl ofeach denatured, diluted SDA reaction was added to each of three wells.In one well the SDA reaction products were hybridized with the IS6110capture and detector probe set, in the second well the SDA reactionproducts were hybridized with the 16S capture and detector probe set andin the third well the SDA reaction products were hybridized with theinternal control capture and detector probe set. Immediately afteraddition of the sample to the well, 50 μl/well of hybridization mix (100mM sodium phosphate, 0.02% BSA, 1.8M NaCl, 0.2% NaN₃, 0.1 mM ZnCl₂, 20μg/ml sheared salmon sperm DNA, pH 7.0, capture and detector probes) wasadded. The plate was covered and incubated for 45 min. at 37° C. Threestringency washes (300 μl/well--10 mM Tris pH 7.5, 0.1% w/v BSA, 0.01%v/v NONIDET P-40, 250 mM NaCl) were performed at room temperature. Eachwash was allowed to remain in the wells for 1 min. before removing.LUMIPHOS 530 AP substrate (100 μl/well) was added and the plates werecovered and incubated for 30 min. at 37° C. Luminescence (Relative LightUnits--RLU) was read on a microtiter plate luminometer (Labsystems,Research Triangle Park, N.C.) at 37° C., using a 2 second/wellintegration time.

The results, in RLU, are shown in the following table. "Over" indicatesan RLU reading above the maximum recordable by the instrument.

    ______________________________________                                                   SPECIES-    GENUS-                                                            SPECIFIC    SPECIFIC- INTERNAL                                     GENOMES    (IS6110)    (16S)     CONTROL                                      ______________________________________                                         0          11          12       8515                                          5          300         68       Over                                         10          945        164       7744                                         20         3157        615       Over                                         40         3618        694       14118                                        80         6890        1367      Over                                         Heat Killed                                                                                5          18         3                                          ______________________________________                                    

The assay detected as little as five genomic copies of 16S rDNA and 5genomic copies of IS6110 from M.tb above background. Amplificationactivity, as evidenced by the high levels of amplification of theinternal control sequence, was high in each sample, i.e., the sampleswere not inhibitory to the amplification reaction.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGAATAGTCG GTTACTTGTTGACGGCGTACTCGACC37                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTCGCGTTGTTCACTG AGATCCCCT25                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGGACCCGCCAAC 13                                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGCTGAACCGGAT 13                                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTCTATAGTCGGTTACTTGTTGACGTCGCG TTGTTC36                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGCGTACTCGACCACGCTCACAGTTA 26                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGGAATTACTGGG 13                                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGTCTGCCCGTATC 14                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCCGTATGGTGGATA 15                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCCGTGAGATTTCAC 15                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCTGTGAGTTTTCAC 15                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GACGGCGTACTCGACCAGCGACGATGTCTGAGGCAACTAGCAAAGCTGAACAACGCGAC 59                (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCTGAAAGACGTTAT15                                                             (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCACCATACGGATAGT16                                                             (2) INFORMATION FOR SEQ ID NO:15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GCTTTGCTAGTTGCC15                                                             (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TCAGACATCGTCGCT15                                                             (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ACTGTGAGCGTGGTC15                                                             (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AAATCTCACGGCTTA15                                                             (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AAAACTCACAGCTTA15                                                         

What is claimed is:
 1. A method for simultaneously amplifying multipleMycobacterium target sequences in a sample comprising:a) hybridizing afirst primer consisting of SEQ ID NO: 1 to the target sequences,extending the first primer with polymerase to produce a first extensionproduct and displacing the first extension product; b) hybridizing asecond primer consisting of SEQ ID NO: 2 to the first extension product,extending the second primer to produce a second extension product anddisplacing the second extension product; c) hybridizing a third primerconsisting of SEQ ID NO: 5 to the target sequences, extending the thirdprimer with polymerase to produce a third extension product anddisplacing the third extension product; d) hybridizing a fourth primerconsisting of SEQ ID NO: 6 to the third extension product, extending thefourth primer with polymerase to produce a fourth extension product anddisplacing the fourth extension product, and; e) simultaneouslyamplifying the second and fourth extension products in a StrandDisplacement Amplification reaction using; SEQ ID NO: 1 and SEQ ID NO: 5as amplification primers.
 2. The method of claim 1 further comprisingdetecting amplification products of step (e) as an indication ofpresence or absence of Mycobacterium tuberculosis complex species orspecies of the genus Mycobacterium in the sample.
 3. The method of claim2 wherein the first, second, third or fourth extension product isdisplaced by extension of a bumper primer.
 4. The method of claim 3wherein the bumper primer consists of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 7 or SEQ ID NO:
 8. 5. The method of claim 3 wherein the first andthird primers are present in approximately 10-fold excess over the thirdand fourth primers.
 6. The method of claim 2 wherein the second andfourth extension products are amplified using a restriction enzymeselected from the group consisting of HincII, HindII, AvaI, NciI andFnu4HI.
 7. The method of claim 2 wherein the amplification products aredetected by hybridization and extension of a ³² P-labeled primer.
 8. Themethod of claims 1 or 2 further comprising the steps of:a) hybridizingthe second primer to a second strand of the target sequences, extendingthe second primer with polymerase to produce a fifth extension productand displacing the fifth extension product; b) hybridizing the firstprimer to the fifth extension product, extending the first primer withpolymerase to produce a sixth extension product and displacing the sixthextension product; c) hybridizing the fourth primer to a second strandof the target sequences, extending the fourth primer with polymerase toproduce a seventh extension produce and displacing the seventh extensionproduct; d) hybridizing the third primer to the seventh extensionproduct, extending the third primer with polymerase to produce an eighthextension product and displacing the eighth extension product, and; e)simultaneously amplifying the sixth and eighth extension products in aStrand Displacement Amplification reaction using SEQ ID NO: 1 and SEQ IDNO: 5 as amplification primers.
 9. A method for simultaneouslyamplifying multiple Mycobacterium target sequences and an internalcontrol sequence in a sample comprising:a) adding an internal controlsequence consisting of SEQ ID NO: 12 to the sample; b) hybridizing afirst primer consisting of SEQ ID NO: 1 to the target sequences,extending the first primer with polymerase to produce a first extensionproduct and displacing the first extension product; b) hybridizing asecond primer consisting of SEQ ID NO: 2 to the first extension product,extending the second primer to produce a second extension product anddisplacing the second extension product; c) hybridizing a third primerconsisting of SEQ ID NO: 5 to the target sequences, extending the thirdprimer with polymerase to produce a third extension product anddisplacing the third extension product; d) hybridizing a fourth primerconsisting of SEQ ID NO: 6 to the third extension product, extending thefourth primer with polymerase to produce a fourth extension product anddisplacing the fourth extension product, and; e) simultaneouslyamplifying the second and fourth extension products and the internalcontrol sequence in a Strand Displacement Amplification reaction usingSEQ ID NO: 1 and SEQ ID NO: 5 as amplification primers.
 10. The methodof claim 9 further comprising detecting the amplification products ofstep (e) as an indication of presence or absence of Mycobacteriumtuberculosis complex species or species of the genus Mycobacterium inthe sample.
 11. The method of claim 10 wherein the first, second, thirdor fourth extension product is displaced by extension of a bumperprimer.
 12. The method of claim 11 wherein the bumper primer consists ofSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO:
 8. 13. The methodof claim 11 wherein the first and third primers are present inapproximately 10-fold excess over the third and fourth primers.
 14. Themethod of claim 10 wherein the second and fourth extension products areamplified using a restriction enzyme selected from the group consistingof HincII, HindII, AvaI, NciI and Fnu4HI.
 15. The method of claim 10wherein the amplification products are detected in a chemiluminescentassay by hybridization to an alkaline phosphatase-labeled detectorprobe.
 16. The method of claims 9 or 10 further comprising the stepsof:a) hybridizing the second primer to a second strand of the targetsequences, extending the second primer with polymerase to produce afifth extension product and displacing the fifth extension product; b)hybridizing the first primer to the fifth extension product, extendingthe first primer with polymerase to produce a sixth extension productand displacing the sixth extension product; c) hybridizing the fourthprimer to a second strand of the target sequences, extending the fourthprimer with polymerase to produce a seventh extension produce anddisplacing the seventh extension product; d) hybridizing the thirdprimer to the seventh extension product, extending the third primer withpolymerase to produce an eighth extension product and displacing theeighth extension product, and; e) simultaneously amplifying the sixthand eighth extension products in a Strand Displacement Amplificationreaction using SEQ ID NO: 1 and SEQ ID NO: 5 as amplification primers.17. An oligonucleotide consisting of a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6.18. An oligonucleotide consisting of SEQ ID NO:
 12. 19. Anoligonucleotide consisting of a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:
 19. 20. Theoligonucleotide of claim 19 which is conjugated to a label.
 21. Theoligonucleotide of claim 20 wherein the label is alkaline phosphatase orbiotin.
 22. A method for genus-specific amplification of Mycobacteriumtarget sequences in a sample comprising:a) hybridizing a first primerconsisting of SEQ ID NO: 5 to the target sequences, extending the firstprimer with polymerase to produce a first extension product anddisplacing the first extension product; b) hybridizing a second primerconsisting of SEQ ID NO: 6 to the first extension product, extending thesecond primer to produce a second extension product and displacing thesecond extension product, and; e) amplifying the second extensionproduct in a Strand Displacement Amplification reaction using SEQ ID NO:1 and SEQ ID NO: 5 as amplification primers.
 23. The method of claim 22further comprising simultaneous amplification of an internal controlsequence consisting of SEQ ID NO: 12 and the second extension product.24. The method of claim 22 further comprising the steps of:a)hybridizing the second primer to a second strand of the targetsequences, extending the second primer with polymerase to produce athird extension product and displacing the third extension product; b)hybridizing the first primer to the third extension product, extendingthe first primer with polymerase to produce a fourth extension productand displacing the fourth extension product, and; c) amplifying thefourth extension product in a Strand Displacement Amplification reactionusing SEQ ID NO: 1 and SEQ ID NO: 5 as amplification primers.
 25. A kitfor amplifying Mycobacterium target sequences comprising:a) a firstoligonucleotide consisting of SEQ ID NO: 1, a second oligonucleotideconsisting of SEQ ID NO: 2, a third oligonucleotide consisting of SEQ IDNO: 5 and a fourth oligonucleotide consisting of SEQ ID NO: 6, and; b)reagents for hybridizing the first, second, third and fourtholigonucleotides to the Mycobacterium target sequences, extending theoligonucleotides and displacing the oligonucleotides from the targetsequences in an amplification reaction.
 26. A kit for genus-specificamplification of Mycobacterium target sequences comprising:a) a firstoligonuclectide consisting of SEQ ID NO: 5 and a second oligonucleotideconsisting of SEQ ID NO: 6, and b) reagents for hybridizing the firstand second oligonucleotides to the Mycobacterium target sequences,extending the oligonucleotides and displacing the oligonucleotides fromthe target sequences in an amplification reaction.
 27. The kit of claims25 or 26 further comprising an oligonucleotide consisting of SEQ ID NO:12 as an internal control sequence for the amplification.
 28. The kit ofclaim 27 further comprising an oligonucleotide for detection ofamplified target sequences.
 29. The kit of claim 28 wherein theoligonucleotide for detection of amplified target sequences comprises anucleotide sequence consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18 or SEQ ID NO:
 19. 30. The kit of claim 29wherein the oligonucleotide for detection of amplified target sequencesis conjugated to a label.
 31. The kit of claim 30 wherein the label isalkaline phosphatase or biotin.